Supplemental Information. The E2F functional analog SBF recruits the Rpd3(L) HDAC, via Whi5 and Stb1,

Transcription

1 The E2F functional analog SBF recruits the Rpd3(L) HDAC, via Whi5 and Stb1, and the FACT chromatin reorganizer, to yeast G1 cyclin promoters Shinya Takahata, Yaxin Yu, and David J. Stillman Supplemental Figure S1. Rpd3(L) binding to promoters can be affected by a stb1 mutation....2 Supplemental Figure S2. SBF binding to CLN1 and CLN2 is not affected by stb1 or whi5 mutations....3 Supplemental Figure S3. Effect of whi5 and stb1 mutations on MBF gene expression....4 Supplemental Figure S4. Whi5 interacts only with SBF....5 Supplemental Figure S5. Whi5 is required for Cdc28 recruitment to SBF and MBF dependent promoters....6 Supplemental Figure S6. CLN2 expression and Gcn5 binding delayed in pob3 and swi6 mutants....7 Supplemental Figure S7. An rme1 mutation affects CLN2 expression....9 Supplemental Figure S8. A swi6 mutation affects FACT binding and nucleosome eviction at SBF- and MBF-dependent promoters Supplemental Figure S9. whi5 stb1 double mutants show additive growth defect at 37 C. 11 Supplemental Table S1. Strain List Supplemental Table S2. Oligonucleotide List....15

2 Supplemental Figure S1. Rpd3(L) binding to promoters can be affected by a stb1 mutation. Strains DY12247 (GALp::CDC20 SDS3-Myc), DY13500 (GALp::CDC20 SDS3-Myc stb1), and DY13498 (GALp::CDC20 SDS3-Myc whi5) were synchronized and ChIP experiments preformed to measure Rpd3(L) binding at CLN1, CLN2, CDC21, and CDC6. 2

3 Supplemental Figure S2. SBF binding to CLN1 and CLN2 is not affected by stb1 or whi5 mutations. SBF binding to promoters was measured in log phase wild type (DY6241 SWI4-Myc), stb1 (DY13998 SWI4-Myc stb1), whi5 (DY14000 SWI4-Myc whi5), and stb1 whi5 (DY14002 SWI4-Myc stb1 whi5), DY150 was used as the untagged control. 3

4 Supplemental Figure S3. Effect of whi5 and stb1 mutations on MBF gene expression. CDC21 and POL1 expression is reduced in stb1 mutants but less so in stb1 whi5 double mutants. CYC1 expression is not affected by whi5 or stb1 mutations. RNA was isolated from log phase wild type (DY150), whi5 (DY10474), stb1 (DY13454), and stb1 whi5 (DY13494), strains, and mrna levels measured by RT-qPCR. 4

5 Supplemental Figure S4. Whi5 interacts only with SBF. A. Whi5 immunoprecipitates with the Swi4 subunit of SBF. Extracts prepared from strains DY14027 (WHI5-Flag), DY6241 (SWI4-Myc), and DY14057 (WHI5-Flag SWI4- Myc), were immunoprecipitated with either anti-myc or anti-flag antibody and analyzed on western blots. B. Whi5 does not immunoprecipitate with the Mbp1 subunit of MBF. Extracts prepared from strains DY14027 (WHI5-Flag), DY8340 (MBP1-Myc), and DY14058 (WHI5-Flag MBP1-Myc), were immunoprecipitated with either anti-myc or anti-flag antibody and analyzed on western blots. 5

6 Supplemental Figure S5. Whi5 is required for Cdc28 recruitment to SBF and MBF dependent promoters. Cdc28 binding to promoters was measured in log phase wild type (DY13020 CDC28- Myc), swi6 (DY13023 CDC28-Myc swi6), stb1 (DY13029 CDC28-Myc stb1), whi5 (DY13031 CDC28-Myc whi5), and stb1 whi5 (DY13023 CDC28-Myc stb1 whi5) cells. DY150 was used as the untagged control. 6

7 Supplemental Figure S6. CLN2 expression and Gcn5 binding delayed in pob3 and swi6 mutants. A. In swi6 or pob3 mutants CLN2 RNA expression is delayed. RNA was isolated from synchronized strains DY12752 (GALp::CDC20 GCN5-Myc), DY12750 (GALp::CDC20 GCN5-Myc pob3), and DY13373 (GALp::CDC20 GCN5-Myc swi6) and HO and CLN2 7

8 mrna levels measured by RT-qPCR. Note that the CLN2 RNA time course from wild type cells is also displayed in Fig 1A of the main text. B. In swi6 or pob3 mutants HO RNA is eliminated. RNA was isolated from synchronized strains DY12752 (GALp::CDC20 GCN5-Myc), DY12750 (GALp::CDC20 GCN5-Myc pob3), and DY13373 (GALp::CDC20 GCN5-Myc swi6) and HO and CLN2 mrna levels measured by RT-qPCR. C. Gcn5 binding to CLN2 is delayed in swi6 or pob3 mutants. Strains DY12752 (GALp::CDC20 GCN5-Myc), DY12750 (GALp::CDC20 GCN5-Myc pob3), and DY13373 (GALp::CDC20 GCN5-Myc swi6) were synchronized and Gcn5 binding to CLN2 was determined by ChIP. 8

9 Supplemental Figure S7. An rme1 mutation affects CLN2 expression. CLN2 expression is additvely reduced in swi6 rme1 double mutants and in pob3 rme1 double mutants. HO is expression is not additively reduced in the double mutants. PIR1 epxression is not affected by rme1, swi6 or pob3 mutations. RNA was isolated from log phase wild type (DY150), rme1 (DY13867), swi6 (DY13357), swi6 rme1 (DY13905), pob3 (DY7379), and pob3 rme1 (DY13909), strains, and mrna levels measured by RTqPCR. 9

10 Supplemental Figure S8. A swi6 mutation affects FACT binding and nucleosome eviction at SBF- and MBF-dependent promoters. Strain DY13373 (GALp::CDC20 swi6) was synchronized and samples taken for FACT (Spt16) ChIP, histone ChIP, and mrna measurements for (A) HO URS2, (B) PIR1, (C) CLN1, (D) CLN2, (D) CDC21, and (F) POL1. 10

11 Supplemental Figure S9. whi5 stb1 double mutants show additive growth defect at 37 C. Serial dilutions of strains DY150 (wild type), DY10474 (whi5), DY13454 (stb1), DY13494 (stb1 whi5), were incubated at various temperatures. Photographs were taken after 1 and 2 days growth. 11

12 Supplemental Table S1. Strain List. DY150 DY6241 DY6669 DY7379 DY7815 DY8340 DY9558 MATa ade2 can1 his3 leu2 trp1 ura3 MATa SWI4-Myc::TRP1 ade2 can1 his3 leu2 trp1 ura3 MATa GALp::CDC20::ADE2 ade2 can1 his3 leu2 trp1 ura3 MATa pob3(l78r) ade2 can1 his3 leu2 lys2 met15 trp1 ura3 MATa spt16-11 ade2 can1 his3 leu2 lys2 trp1 ura3 MATa MBP1-Myc::TRP1 ade2 can1 his3 leu2 trp1 ura3 MATα whi5::kanmx ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY10474 MATa whi5::kanmx ade2 can1 his3 leu2 lys2 trp1 ura3 DY11246 MATa GALp::CDC20::ADE2 pob3(l78r) ade2 can1 his3 leu2 lys2 trp1 ura3 DY11982 MATa cdc28-13 whi5::kanmx ade2 can1 his3 leu2 trp1 ura3 DY12236 MATa pob3(q308k)::kanmx ade2 can1 his3 leu2 lys2 trp1 ura3 DY12247 MATa SDS3-Myc::His3MX GALp::CDC20::ADE2 ade2 can1 his3 leu2 lys2 trp1 ura3 DY12248 MATα SDS3-Myc::His3MX GALp::CDC20::ADE2 ade2 can1 his3 leu2 lys2 trp1 ura3 DY12750 MATa GCN5-Myc::KanMX GALp::CDC20::ADE2 pob3(l78r) ade2 can1 his3 leu2 lys2 trp1 ura3 DY12752 MATa GCN5-Myc::KanMX GALp::CDC20::ADE2 ade2 can1 his3 leu2 lys2 trp1 ura3 DY12794 MATa SWI4-Myc::TRP1 GALp::CDC20::ADE2 ade2 can1 his3 leu2 trp1 ura3 DY12828 MATa SDS3-Myc::His3MX GALp::CDC20::ADE2 swi6::trp1 ade2 can1 his3 leu2 trp1 ura3 12

13 DY12914 MATa GALp::CDC20::ADE2 asf1::trp1 ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY13020 MATa CDC28-Myc::KanMX ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY13357 MATa swi6::trp1 ade2 can1 his3 leu2 trp1 ura3 DY13369 MATa GCN5-Myc::KanMX GALp::CDC20::ADE2 swi6::trp1 ade2 can1 his3 leu2 lys2 trp1 ura3 DY13373 MATa GALp::CDC20::ADE2 swi6::trp1 ade2 can1 his3 leu2 lys2 trp1 ura3 DY13454 MATa stb1::trp1 ade2 can1 his3 leu2 lys2 trp1 ura3 DY13494 MATa stb1::trp1 whi5::kanmx ade2 can1 his3 leu2 trp1 ura3 DY13496 MATa GALp::CDC20::ADE2 stb1::trp1 whi5::kanmx ade2 can1 his3 leu2 trp1 ura3 DY13498 MATa SDS3-Myc::His3MX GALp::CDC20::ADE2 whi5::kanmx ade2 can1 his3 leu2 trp1 ura3 DY13502 MATα SDS3-Myc::His3MX GALp::CDC20::ADE2 stb1::trp1 whi5::kanmx ade2 can1 his3 leu2 trp1 ura3 DY13541 MATa cdc28-13 stb1::trp1 ade2 can1 his3 leu2 trp1 ura3 DY13543 MATa cdc28-13 stb1::trp1 whi5::kanmx ade2 can1 his3 leu2 trp1 ura3 DY13640 MATa whi5::hphmx stb1::trp1 ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY13696 MATa pob3(q308k)::kanmx stb1::trp1 ade2 can1 his3 leu2 lys2 trp1 ura3 DY13698 MATa pob3(q308k)::kanmx whi5::hphmx ade2 can1 his3 leu2 lys2 trp1 ura3 DY13700 MATa pob3(q308k)::kanmx whi5::hphmx stb1::trp1 ade2 can1 his3 leu2 lys2 trp1 ura3 DY13702 MATa pob3(l78r) whi5::hphmx ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY13704 MATa pob3(l78r) stb1::trp1 ade2 can1 his3 leu2 lys2 met15 trp1 ura3 13

14 DY13706 MATa pob3(l78r) stb1::trp1 whi5::hphmx ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY13714 MATa spt16-11 stb1::trp1 ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY13716 MATa spt16-11 whi5::hphmx ade2 can1 his3 leu2 lys2 trp1 ura3 DY13718 MATa spt16-11 stb1::trp1 whi5::hphmx ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY13723 MATa CDC28-Myc::KanMX swi6::trp1 ade2 can1 his3 leu2 lys2 trp1 ura3 DY13729 MATa CDC28-Myc::KanMX stb1::trp1 ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY13731 MATa CDC28-Myc::KanMX whi5::hphmx ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY13733 MATa CDC28-Myc::KanMX stb1::trp1 whi5::hphmx ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY13867 MATa rme1::kanmx ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY13905 MATa rme1::kanmx swi6::trp1 ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY13909 MATa rme1::kanmx pob3(l78r) ade2 can1 his3 leu2 lys2 met15 trp1 ura3 DY13998 MATa SWI4-Myc::TRP1 stb1::hphmx ade2 can1 his3 leu2 trp1 ura3 DY14000 MATa SWI4-Myc::TRP1 whi5::kanmx ade2 can1 his3 leu2 trp1 ura3 DY14002 MATa SWI4-Myc::TRP1 stb1::hphmx whi5::kanmx ade2 can1 his3 leu2 trp1 ura3 DY14024 MATa pob3(l78r) swi6::trp1 ade2 can1 his3 leu2 lys2 trp1 ura3 + YCp(URA3)- POB3 DY14027 MATa WHI5-Flag::His3MX ade2 can1 his3 leu2 trp1 ura3 DY14057 MATa SWI4-Myc::TRP1 WHI5-Flag::His3MX ade2 can1 his3 leu2 trp1 ura3 DY14058 MATa MBP1-Myc::TRP1 WHI5-Flag::His3MX ade2 can1 his3 leu2 trp1 ura3 14

15 Supplemental Table S2. Oligonucleotide List. RT-PCR oligos F1066 HO AAATGGAGCGCTCTAAAGGAGAA F1067 HO CTAACCACAGACCAAGCATCCAA F2171 CLN1 CAAACACTCAAACGCAGGTATTCAG F2172 CLN1 GGTCTATTGTAGAGGCCAGTTGCAG F2043 CLN2 AATTCTTGATTGATGTATCCGCGTGG F2044 CLN2 GTTAGGAATGGAAACAATGCCGTTCA F2302 CDC21 TGTTGCTTTTTTGACCTTCT F2303 CDC21 GAAGCTCTCTCCTTGATACTT F2350 POL1 TAAAGGGAGCAGATTTCAATTC F2351 POL1 GGTGAATCCAACGAGATATTT F1719 PIR1 CCACCACCAATACTACTGTTGCTC F1720 PIR1 ACCTTTACCGTCAGTCAGGATACC F2145 CYC1 TTCTGCTAAGAAAGGTGCTAC F2146 CYC1 TCTTTGGGTTAGTCAAGTACT F2173 RDN25 (control) CGTTCCTTGTCTATGTTCCTTG F2174 RDN25 (control) CACTGTACTTGTTCGCTATCG ChIP oligos F1095 HO URS2 GGCAAACCTAATGTGACCGT F1096 HO URS2 GGCAAACCTAATGTGACCGT F2300 CDC6 TTTGTTTTCCCTGAATTACCC F2301 CDC6 AAATAGTGCAGCGCAGTG F1605 CDC20 TCCTTCACTTCCTTTATGTCC F1606 CDC20 TCGCAAAGCAGATTAGTCCT F2302 CDC21 TGTTGCTTTTTTGACCTTCT F2303 CDC21 GAAGCTCTCTCCTTGATACTT 15

16 F2127 CLN1 GACGATCCACTAGCGACTTCT F2128 CLN1 CACGCTGCCTTTACTCCATTC F996 CLN2 GTTATCAATTCATGCGCGCT F997 CLN2 AGATCAACATTTCGCAGGTT F1295 CTS1 ACCAGCCTCGCCTTAACAGA F1296 CTS1 GACCAATGCCTATGAAGCCAA F1759 PIR1 ATAATTCCTCGAAGCCAGAC F1760 PIR1 CTTCTATAATTGGCAGGACATAAA F2298 POL1 TGCCTGCAATCTCTATTCTTC F2299 POL1 TTTCCACTGTTTATTTTATGCCT F1416 Chrom I control GTTTATAGCGGGCATTATGCGTAGATCAG F1417 Chrom I control GTTCCTCTAGAATTTTTCCACTCGCACATTC 16